可控性树突状细胞活疫苗产生抗卵巢上皮性癌免疫应答的实验研究  

Controlled live dendritic cell vaccine mediates potent antitumor immune responses

作　　者：康玉[1] 徐丛剑[1] 刘惜时[1] 张新华[2] 吴超群[3] 钟翠平[2] 顾健人[4] 

机构地区：[1]复旦大学附属妇产科医院妇科,上海200011 [2]上海医学院组胚解剖系 [3]遗传工程国家重点实验室 [4]上海市肿瘤研究所癌基因及相关国家重点实验室

出　　处：《中华妇产科杂志》2004年第10期669-674,i001,共7页Chinese Journal of Obstetrics and Gynecology

基　　金：国家自然科学基金资助项目(30070783);上海市重大科技攻关项目资助(03DZ19234)

摘　　要：目的研究可调控存活状态的树突状细胞(DC)活疫苗在体内产生的抗肿瘤免疫效应。方法(1)将大鼠骨髓来源的DC与转导有自杀基因Ⅰ型单纯疱疹病毒胸苷激酶基因(HSV1TK)的卵巢上皮性癌细胞株NuTu19细胞,以聚乙二醇法融合制成DC活疫苗。以流式细胞仪、共聚焦激光显微镜分析DC活疫苗的生物学特性;RTPCR技术、蛋白印迹法鉴定DC活疫苗中TK基因的表达;四甲基偶氮唑蓝比色法测定更昔洛韦(GCV)对DC活疫苗的杀伤效应。(2)在体内实验中,将大鼠分为活疫苗组、活疫苗+GCV组、死疫苗组、DC+NuTu19/TK组、GCV组和对照组。各组大鼠经二次免疫后,或以乳酸脱氢酶释放法检测脾T淋巴细胞的细胞毒活性,或再给予NuTu19细胞攻击后观察肿瘤的发生与生长情况。活疫苗+GCV组在给予NuTu19细胞攻击后1周开始腹腔注射GCV,将疫苗在体内灭活。结果(1)DC活疫苗高表达表面分子主要组织相容性复合物Ⅱ类分子OX6、共刺激分子B72、整合素OX62和细胞间黏附分子ICAM1,其阳性率分别为(876±34)%、(711±93)%、(680±74)%和(771±20)%;DC与NuTu19/TK细胞的融合率为(23±14)%;DC活疫苗中TK基因呈阳性表达。(2)活疫苗组大鼠脾T淋巴细胞对NuTu19细胞的杀伤活性为(618±83)%,明显高于死疫苗组的(260±38)%(P<005)。与对照组相比。Objective To investigate the efficiency of antitumor immune responses induced by a controlled live dendritic cell(DC)vaccine Methods DC precursors were isolated from Fischer 344 rat bone marrow and cultured with granulocyte macrophage colony stimulating factor and interleukin 4 The rat ovarian tumor cell line NuTu 19 was genetically modified by retroviral mediated suicide gene(HSV 1 TK), and the positive clones were selected using G418 Live DC vaccine was then fused with DC and NuTu 19/TK cell by polyethylene glycol The characteristics of live DC vaccine were assayed with flow cytometry and confocal laser scanning microscopy The specific expression of HSV 1 TK gene in live DC vaccine was evaluated by RT PCR and western blot The sensitivity of live DC vaccine to ganciclovir (GCV) was evaluated by methylthiazoletetrazolium assay In vivo, rats vaccinated twice with live DC vaccine were compared to those vaccinated with killed DC vaccine, unfused DC and NuTu 19/TK cell or phosphate buffered saline Seven days following the last immunization, the rats were sacrificed to test the specific cytotoxic T lymphocyte (CTL) activity by lactate dehydrogenase release assay, or challenged with NuTu 19 and tumor incidence was observed Results The fusion efficiency was approximately (23±14) Live DC vaccine displayed an up regulated expression of major histocompatibility complex (MHC) IIOX6 (87 6±3 4)%, costimulatory molecule B 1 2 (71 1±9 3)%, integrin OX 62 (68 0±7 4)%, and adhesion ICAM 1 (77 1±2 0)%, and specifically expressed HSV 1 TK gene. Our data showed that spleen T lymphocytes from rats vaccinated with live vaccine displayed enhanced CTL aetivity (61 8±8 3)% contrast to that of rats vaccinated with killed vaccines (26 0±3 8)% ( P <0 05). Compared to the control groups, rats immunized with live DC vaccine demonstrated a significant delay in tumor development [(39±8)d vs (70±16)d], reduced tumor incidence (100% vs 80%) and decreased tumor volume [(806±553)mm 3 vs (89±53)mm 3, P <0 05]. Seventy three percent of TK

关 键 词：GCV DC 活疫苗 大鼠 卵巢上皮性癌 体内 HSV 细胞 存活 高表达 

分 类 号：R737.31[医药卫生—肿瘤]